- Email: [email protected]
A versatile thermal biosensor
Sensors and Actuators,
19 (1989)
53
53 - 59
A VERSATILE TIZERMAL BIOSIENSOR XIE Bin and REN Shu
Department of Biomedical Engineering, (People’s Repu biic of China)
Tongii Medical
University,
W&an
(Received 30 June, 1988; in revised form 6 October 1988; accepted 9 November, 1988)
Abstract A newly designed thermal biosensor (TBS) is reported. It is constructed by integrating a temperature sensor with a bioreactor, which can be easily dismantled. Such a design makes the TBS versatile. Both differential and adiabatic techniques are used to improve its precision and resolution. As an example of application, a glucose assay is described: resolution 2 X lo-* mol, linear range 2 X lO‘-8 mol-1.7 X 10d mol, relative error < 6%, lifetime more than one month and sample volume 4 X 10m2 ml.
1. Introduction A specific biochemical reaction always accompanies a process with nonspecific heat change. This phenomenon has attracted the attention of scientists and engineers to the research and development of a thermal biosensor (TBS). Now the TBS has been widely used in many areas of bioanalyses, such as clinical analyses, process control, fermentation analyses, environmental control and ~rn~olo~~l analyses [ 1] _ Up to now, however, all TBSs can merely perform a specific sensor function. Their practical app~cations are restricted because of complicated immobilization methods and their non-versatility. Moreover, interference due to temperature ~u~uations during the process of me~~ment is still a problem to be solved [2]. On account of these facts, a newly designed TBS was developed in our Department. This TBS is constructed by integrating a sensing element with a reaction vessel to reduce the effects of temperature fluctuations. Both differential and adiabatic techniques are used to improve the precision and resolution f33. The bioreactor can be conveniently taken out and replaced. It is possible that the TBS could be used as a versatile biosensor, if different kinds of immobilized membranes made of biomaterials were deposited on the temperature sensor. 0250”6874/89/$3.50
@ Elsevier Sequoia/Printed in The Netherlands
2. Construction Figure 1 shows the TBS’s construction. As may be seen in this Figure, a symme~~ structure is used. The TBS contains two bioreactors, which are situated within a heat insulation column made of polyurethane. One bioreactor functions as a reference vessel, R, and the other as a sample vessel, S. The outer tube of the vessel is firmly fixed to the pol~eth~e eohunn. The space around each bioreactor is evacuated to reduce heat leakage by conduction. Figure 2 shows a detailed drawing of the bioreactor. It includes four parts, i.e., inlet and outlet conduits, a removable rubber stopper, a thickfilm platinum resistor coated with biomaterial immobilized membrane and a silastic tube. Since the conduits and Pt resistor are fixed in the removable rubber stopper, the membrane on the thick-film Pt resistor can be eaisily replaced. The Pt resistor divides each bioreactor into two equal parts. For discontinuous analyses, the upper part is used as a bioreaction bed and the lower part as space for equilib~t~~ the sample t~rn~rat~e. For continuous analyses, a biomat+xM immobilized membrane may be deposited onto both the upper and lower surfaces of the resistor to increase the reactive area. The upper and lower parts are connected by an opening to allow sample flow between them, The advantages of such a design are: (1) reduction in tempolyurethane insulation column /
hole for evacuating: /
vacuum cavity Fig. 1. Schematic vertical cross-section of the versatile TBS.
lead wire Fig. 2. Detailed drawing of the Moreactor (dimensions
in mm).
55
perature disturbance during injection of sample; (2) versatility through de~siting different kinds of membranes on the film resistor; (3) speeding the reaction rate and improving sensitivity due to the larger contact surface between membrane and substrate; (4) reducing heat leakage by means of an adiabatic design; (5) ~rn~~h~g ~~rference due to d~erent~l operation; and (6) ease of cleaning. 3. Experimental In this paper, we have emphasized the design priniciple of a versatile TBS rather than describing the development of a specific biosensor in detail. Here we just give some key procedures about how to get a glucose biosensor on this TBS and how to proceed with measurements. 3. I. ~~~~~i~~~~~u~ Many immobilization methods can be used here [4,5]. We used simple and convenient method to immobilize the glucose oxidase. Figure shows the confiiation of the sensing element of the TBS.
3”
mexnbrane containfna immobilized enmyme
/
silastia. layer \ thick-film Fig. 3. The ~nfi~r~~ion
Pt resistor of the TBS’s sensing elexnent.
~e~e~~s: glucose oxidase (GOD, Sigma Chemical Corn~y~, catalase (CAT, Sigma Chemical Company), albumin (BSA, Shanhai Biochemical Reagent Company). ~u~~~~~~: (1) dissolve 1 g silastic in 30 ml ~~y~of~n; (2) dissolve 1 g cellulose acetate in 20 ml acetone; (3) dissolve 26.3 mg BSA in 0.15 ml phosp~te-buffet saline (PBS), pH 6.8; (4) dissolve 10 mg GOD and 1 mg CAT in 0.27 ml PBS, pH 6.8. Procedure: (1) coat the cleaned film resistor with solution (1) to a thicket of about 0.05 mm, waiting ten minutes for drying; (2) coat the film just obtained with solution (2) to a thickness of about 0.1 mm, waiting ten minutes for drying; (3) a mixed solution, consisting of solutions (3), (4) and 5% glu~~dehyd~ (l.:2:1 by volume) is added to the resistor coating for crosslinking. CAT is used as a stabilizer and BSA provides a mieroenvironment favourable for enzymatic activity.
56
3.2. Measurement The block diagram of the measurement system for the TI3S is shown in Fig. 4.
Fig. 4. The measurement
system of the TBS.
In order to reduce the temperature disturbance during sample injection, in discontinuous measurement, it is necessary to equilibrate the sample temperature before measurement. Initially, the.,sample is injected into the vessel below the Pt resistor and after approximately two hours it is injected into the reaction vessel (see Fig. 2). The disdvantage of this is the time required for temperature equilibration, Obviously, no such problem exists when a continuous-flow method is adopted. In the bridge circuit, each of the two constant resistors is 10 kJ’2and each of the two thick-film Pt resistors (~~~iang Instrumental Company, China) is equal! to 100 G at 20 “C. Such a selection is favourable for interfacing with the amplifier. In addition, a voltage of 3 V was chosen for the power supply to produce a low heat quantity across the Pt resistor (about 10 &vV). Under these conditions the ~nsiti~ty to ~rn~rat~e change is about 100 pV/“c. Moreover, we obtained a gm&er output voltage of 0.1 V/ “C by means of a huh-pr~ision amplifier (Sodom ~niv~sity, China). For an adiabatic system, the temperature change is related to the number of moles of analyte, zt, by:
where AH is the enthalpy of the reaction being measured and C, is the heat capacity of the solution and the surrounding vessel [ 43. If AT, C, and AH are known, then IZcan easily be calculated. According to Fig. 2, the total heat capacity C, of the bioreactor is % = c, f c, + c, = 0.208 J/“C f 0.200 J/“C + 0.533 J/“C - 0.94 J/“C where Cs, Cr and C, are the heat capacities of the sample, Pt resistor and silastic respectively. Here, we have assumed that the heat capacities of the sample and water are approximately equal, and for discontinuous analyses, the bioreactor is only half full of solution. The enthalpy of the catalysis reaction between GOD and glucose is about 80 000 J/mu1 11f . Taking the temperature resolution as O&01%, the resolutiun for glucose can be obtained from the above equation, i.e.,
57
4, R.esuNsand ~s~~ion
A polymer material with a good adsorbability to the resistor and a low affinity for water was sought. A series of materials including polyurethane, polystyrene, polythene, cellulose acetate and silastic was tested. It was found that the adsorbability of silastic is the best among them. The thickness of the membrane affects the properties of the TBS. The appropriate total thickness of the membrane including enzyme membrane, cellulose acetate layer and silastic layer is equal to 0.2 mm. 4.2. ~es~l~~~u~and finear range Using distilled water instead of a sample, the measured temperature ~uct~tion is about 0.0003 “c, A series of glucose determinations has been performed. The resolution for glucose concen~tion rn~~~ernen~ is about 2 X lO_” mol (5 X lO+ mol/l) (see Fig. 5), which agrees with the calculated value given above. Figure 5 shows a linear range of 2 X 10” mol-1.7 X 10d ma1 (5 X lO+ mol/l-4.2 X 10e2 mol/l). Curve A was obtained by adding the same quantity of hydrogen peroxide (0.01% - 0.1%) to each bioreactor to produce complementary oxygen; the quantity of heat produced by the hydrogen peroxide is cancelled out by the differential measurement. 4.3. Reproducibility and lifetime The relative error is less than 6% for ten dedications (see Table 1). The lifetime curve of the TBS is shown in Fig. 6. When it is used once a day, the decrease in enzymatic activity is about 10% after one month. A serum sample from a patient has been tested with this TBS, The result of the glucose con~n~tio~ movement is 160 mg/lOO ml, At the same time the reported clinical result is 161 mg/lOO ml, which agrees well, 3.0 2.5 2.0 x.5
L
0.05
1.4
2.0
4.2
Glucose ( 10-2m31/l)
Fig, 5, The linear range of the TBS,
58 TABLE
1
Reproducibility of the TBS (6.2 x 10v3 mol/l) Number
1
2
3
4
5
6
7
8
9
10
Output (0.001 V)
0.47
0.48
0.46
0.45
0.47
0.48
OS48
0.47
0.48
0.46
i 0
5
10
15
20
25
30 Time(daY)
Fig. 6. The lifetime curve of the TBS.
5. Conclusions The TBS described above. is versatile due to a special design. Primary data suggest the a~cep~bi~ty of such a design. Of course, it remains to be improved; for instance, the inner wall of the column in Fig. 1 should be coated with a silver film in order to prevent heat leakage by radiation. The resolution of 2 X 10d8 mol for glucose and the 4 X low2 ml sample required suggest that this TBS is favourable for mi~oqu~~ty bioanalyses. The principle and technique suggested here are worth continuing study and modification. In this way it should be possible to develop a universal biosensor suitable for all bioanalyses.
Acknowledgement Dr. Bengt Danielsson of the University of Lund, Sweden, presented us with copies of his excellent papers on thermal biosensors for reference. We express our hearty thanks for his help, References 1 B. Danielsson and K. Mosbach, in A. P. F. Turner (ed.), BSusenscmr,Oxford Science Publications, Oxford, 1st edn., 1986, Ch. 29, pp. 582 - 592. 2 B. Danielsson and K. Mosbach, in K. Mosbach (ed.), Methods in Enzymology, Vol. 137, Academic Press, Mew York, 1988, Ch. 16, pp. 181 - 197.
59 3 Ren Shu, Xie Bin and Chang Yu-yi, A newly designed bioactivity monitor, Proc. 2nd MICONEX, Be@ag, China, April 1986, pp. 664 - 669. 4 P. W. Carr and L. D. Bowers, Immobilized Enzymes in Analytical and Clinical Chemistry, Wiley, New York, 1st edn., 1980, pp. 157 - 191, pp. 430 - 431. 5 I. K. Al-Hitti, G. J. Moody and J. D. R. Thomas, Immobilization of membranes for use with electrochemical sensor, J. Biomed. Eng., 6 (1984) 178 - 180.
Biographies XIE Bin was born on September 51958. He received the B.S. degree in electronic engineering from Huazhong University of Science and Technology in 1982, and the M.S. degree in biomedical engineering from Tongji Medical University in 1988. He has been engaged in the research and development of biosensors and biomedical instruments. REN Shu graduated from Wuhan University in 1952. Now he is a professor and director of the Department of Biomedical Engineering and the Department of Environment Monitoring of Tongji Medical University. His current research centres on the development of biosensors and molecular devices.
19 (1989)
53
53 - 59
A VERSATILE TIZERMAL BIOSIENSOR XIE Bin and REN Shu
Department of Biomedical Engineering, (People’s Repu biic of China)
Tongii Medical
University,
W&an
(Received 30 June, 1988; in revised form 6 October 1988; accepted 9 November, 1988)
Abstract A newly designed thermal biosensor (TBS) is reported. It is constructed by integrating a temperature sensor with a bioreactor, which can be easily dismantled. Such a design makes the TBS versatile. Both differential and adiabatic techniques are used to improve its precision and resolution. As an example of application, a glucose assay is described: resolution 2 X lo-* mol, linear range 2 X lO‘-8 mol-1.7 X 10d mol, relative error < 6%, lifetime more than one month and sample volume 4 X 10m2 ml.
1. Introduction A specific biochemical reaction always accompanies a process with nonspecific heat change. This phenomenon has attracted the attention of scientists and engineers to the research and development of a thermal biosensor (TBS). Now the TBS has been widely used in many areas of bioanalyses, such as clinical analyses, process control, fermentation analyses, environmental control and ~rn~olo~~l analyses [ 1] _ Up to now, however, all TBSs can merely perform a specific sensor function. Their practical app~cations are restricted because of complicated immobilization methods and their non-versatility. Moreover, interference due to temperature ~u~uations during the process of me~~ment is still a problem to be solved [2]. On account of these facts, a newly designed TBS was developed in our Department. This TBS is constructed by integrating a sensing element with a reaction vessel to reduce the effects of temperature fluctuations. Both differential and adiabatic techniques are used to improve the precision and resolution f33. The bioreactor can be conveniently taken out and replaced. It is possible that the TBS could be used as a versatile biosensor, if different kinds of immobilized membranes made of biomaterials were deposited on the temperature sensor. 0250”6874/89/$3.50
@ Elsevier Sequoia/Printed in The Netherlands
2. Construction Figure 1 shows the TBS’s construction. As may be seen in this Figure, a symme~~ structure is used. The TBS contains two bioreactors, which are situated within a heat insulation column made of polyurethane. One bioreactor functions as a reference vessel, R, and the other as a sample vessel, S. The outer tube of the vessel is firmly fixed to the pol~eth~e eohunn. The space around each bioreactor is evacuated to reduce heat leakage by conduction. Figure 2 shows a detailed drawing of the bioreactor. It includes four parts, i.e., inlet and outlet conduits, a removable rubber stopper, a thickfilm platinum resistor coated with biomaterial immobilized membrane and a silastic tube. Since the conduits and Pt resistor are fixed in the removable rubber stopper, the membrane on the thick-film Pt resistor can be eaisily replaced. The Pt resistor divides each bioreactor into two equal parts. For discontinuous analyses, the upper part is used as a bioreaction bed and the lower part as space for equilib~t~~ the sample t~rn~rat~e. For continuous analyses, a biomat+xM immobilized membrane may be deposited onto both the upper and lower surfaces of the resistor to increase the reactive area. The upper and lower parts are connected by an opening to allow sample flow between them, The advantages of such a design are: (1) reduction in tempolyurethane insulation column /
hole for evacuating: /
vacuum cavity Fig. 1. Schematic vertical cross-section of the versatile TBS.
lead wire Fig. 2. Detailed drawing of the Moreactor (dimensions
in mm).
55
perature disturbance during injection of sample; (2) versatility through de~siting different kinds of membranes on the film resistor; (3) speeding the reaction rate and improving sensitivity due to the larger contact surface between membrane and substrate; (4) reducing heat leakage by means of an adiabatic design; (5) ~rn~~h~g ~~rference due to d~erent~l operation; and (6) ease of cleaning. 3. Experimental In this paper, we have emphasized the design priniciple of a versatile TBS rather than describing the development of a specific biosensor in detail. Here we just give some key procedures about how to get a glucose biosensor on this TBS and how to proceed with measurements. 3. I. ~~~~~i~~~~~u~ Many immobilization methods can be used here [4,5]. We used simple and convenient method to immobilize the glucose oxidase. Figure shows the confiiation of the sensing element of the TBS.
3”
mexnbrane containfna immobilized enmyme
/
silastia. layer \ thick-film Fig. 3. The ~nfi~r~~ion
Pt resistor of the TBS’s sensing elexnent.
~e~e~~s: glucose oxidase (GOD, Sigma Chemical Corn~y~, catalase (CAT, Sigma Chemical Company), albumin (BSA, Shanhai Biochemical Reagent Company). ~u~~~~~~: (1) dissolve 1 g silastic in 30 ml ~~y~of~n; (2) dissolve 1 g cellulose acetate in 20 ml acetone; (3) dissolve 26.3 mg BSA in 0.15 ml phosp~te-buffet saline (PBS), pH 6.8; (4) dissolve 10 mg GOD and 1 mg CAT in 0.27 ml PBS, pH 6.8. Procedure: (1) coat the cleaned film resistor with solution (1) to a thicket of about 0.05 mm, waiting ten minutes for drying; (2) coat the film just obtained with solution (2) to a thickness of about 0.1 mm, waiting ten minutes for drying; (3) a mixed solution, consisting of solutions (3), (4) and 5% glu~~dehyd~ (l.:2:1 by volume) is added to the resistor coating for crosslinking. CAT is used as a stabilizer and BSA provides a mieroenvironment favourable for enzymatic activity.
56
3.2. Measurement The block diagram of the measurement system for the TI3S is shown in Fig. 4.
Fig. 4. The measurement
system of the TBS.
In order to reduce the temperature disturbance during sample injection, in discontinuous measurement, it is necessary to equilibrate the sample temperature before measurement. Initially, the.,sample is injected into the vessel below the Pt resistor and after approximately two hours it is injected into the reaction vessel (see Fig. 2). The disdvantage of this is the time required for temperature equilibration, Obviously, no such problem exists when a continuous-flow method is adopted. In the bridge circuit, each of the two constant resistors is 10 kJ’2and each of the two thick-film Pt resistors (~~~iang Instrumental Company, China) is equal! to 100 G at 20 “C. Such a selection is favourable for interfacing with the amplifier. In addition, a voltage of 3 V was chosen for the power supply to produce a low heat quantity across the Pt resistor (about 10 &vV). Under these conditions the ~nsiti~ty to ~rn~rat~e change is about 100 pV/“c. Moreover, we obtained a gm&er output voltage of 0.1 V/ “C by means of a huh-pr~ision amplifier (Sodom ~niv~sity, China). For an adiabatic system, the temperature change is related to the number of moles of analyte, zt, by:
where AH is the enthalpy of the reaction being measured and C, is the heat capacity of the solution and the surrounding vessel [ 43. If AT, C, and AH are known, then IZcan easily be calculated. According to Fig. 2, the total heat capacity C, of the bioreactor is % = c, f c, + c, = 0.208 J/“C f 0.200 J/“C + 0.533 J/“C - 0.94 J/“C where Cs, Cr and C, are the heat capacities of the sample, Pt resistor and silastic respectively. Here, we have assumed that the heat capacities of the sample and water are approximately equal, and for discontinuous analyses, the bioreactor is only half full of solution. The enthalpy of the catalysis reaction between GOD and glucose is about 80 000 J/mu1 11f . Taking the temperature resolution as O&01%, the resolutiun for glucose can be obtained from the above equation, i.e.,
57
4, R.esuNsand ~s~~ion
A polymer material with a good adsorbability to the resistor and a low affinity for water was sought. A series of materials including polyurethane, polystyrene, polythene, cellulose acetate and silastic was tested. It was found that the adsorbability of silastic is the best among them. The thickness of the membrane affects the properties of the TBS. The appropriate total thickness of the membrane including enzyme membrane, cellulose acetate layer and silastic layer is equal to 0.2 mm. 4.2. ~es~l~~~u~and finear range Using distilled water instead of a sample, the measured temperature ~uct~tion is about 0.0003 “c, A series of glucose determinations has been performed. The resolution for glucose concen~tion rn~~~ernen~ is about 2 X lO_” mol (5 X lO+ mol/l) (see Fig. 5), which agrees with the calculated value given above. Figure 5 shows a linear range of 2 X 10” mol-1.7 X 10d ma1 (5 X lO+ mol/l-4.2 X 10e2 mol/l). Curve A was obtained by adding the same quantity of hydrogen peroxide (0.01% - 0.1%) to each bioreactor to produce complementary oxygen; the quantity of heat produced by the hydrogen peroxide is cancelled out by the differential measurement. 4.3. Reproducibility and lifetime The relative error is less than 6% for ten dedications (see Table 1). The lifetime curve of the TBS is shown in Fig. 6. When it is used once a day, the decrease in enzymatic activity is about 10% after one month. A serum sample from a patient has been tested with this TBS, The result of the glucose con~n~tio~ movement is 160 mg/lOO ml, At the same time the reported clinical result is 161 mg/lOO ml, which agrees well, 3.0 2.5 2.0 x.5
L
0.05
1.4
2.0
4.2
Glucose ( 10-2m31/l)
Fig, 5, The linear range of the TBS,
58 TABLE
1
Reproducibility of the TBS (6.2 x 10v3 mol/l) Number
1
2
3
4
5
6
7
8
9
10
Output (0.001 V)
0.47
0.48
0.46
0.45
0.47
0.48
OS48
0.47
0.48
0.46
i 0
5
10
15
20
25
30 Time(daY)
Fig. 6. The lifetime curve of the TBS.
5. Conclusions The TBS described above. is versatile due to a special design. Primary data suggest the a~cep~bi~ty of such a design. Of course, it remains to be improved; for instance, the inner wall of the column in Fig. 1 should be coated with a silver film in order to prevent heat leakage by radiation. The resolution of 2 X 10d8 mol for glucose and the 4 X low2 ml sample required suggest that this TBS is favourable for mi~oqu~~ty bioanalyses. The principle and technique suggested here are worth continuing study and modification. In this way it should be possible to develop a universal biosensor suitable for all bioanalyses.
Acknowledgement Dr. Bengt Danielsson of the University of Lund, Sweden, presented us with copies of his excellent papers on thermal biosensors for reference. We express our hearty thanks for his help, References 1 B. Danielsson and K. Mosbach, in A. P. F. Turner (ed.), BSusenscmr,Oxford Science Publications, Oxford, 1st edn., 1986, Ch. 29, pp. 582 - 592. 2 B. Danielsson and K. Mosbach, in K. Mosbach (ed.), Methods in Enzymology, Vol. 137, Academic Press, Mew York, 1988, Ch. 16, pp. 181 - 197.
59 3 Ren Shu, Xie Bin and Chang Yu-yi, A newly designed bioactivity monitor, Proc. 2nd MICONEX, Be@ag, China, April 1986, pp. 664 - 669. 4 P. W. Carr and L. D. Bowers, Immobilized Enzymes in Analytical and Clinical Chemistry, Wiley, New York, 1st edn., 1980, pp. 157 - 191, pp. 430 - 431. 5 I. K. Al-Hitti, G. J. Moody and J. D. R. Thomas, Immobilization of membranes for use with electrochemical sensor, J. Biomed. Eng., 6 (1984) 178 - 180.
Biographies XIE Bin was born on September 51958. He received the B.S. degree in electronic engineering from Huazhong University of Science and Technology in 1982, and the M.S. degree in biomedical engineering from Tongji Medical University in 1988. He has been engaged in the research and development of biosensors and biomedical instruments. REN Shu graduated from Wuhan University in 1952. Now he is a professor and director of the Department of Biomedical Engineering and the Department of Environment Monitoring of Tongji Medical University. His current research centres on the development of biosensors and molecular devices.